DNA sequencing 2 & 3 Flashcards
Illumnia(Solexa)
Prepare ________________ linked to _________
Attach ___________ to ____________ via _______
__________ by “bridge PCR”
__________ using dna template which is
libraries of DNA fragments, adaptors (as short sequence tags)
single stranded DNA fragments, solid phase, adapter sequence
Clonal amplification
sequence by synthesis, anchored to the solid phase
Illumina sequencing by synthesis
_________________ are used as templates
In each cycle, only ___________________ is added, since the __________ is chemically blocked
An image is taken to ____________
The 3’ OH blocking is _________(________ ______), allowing ______________
“Clustered” clonal DNA fragments
one base carrying a base-unique fluorescent label, 3’ OH group
identify the nucleotide incorporated
removed, reversible, terminator, another round of synthesis
ION TORRENT (Personal Genome Machine)
___________ generated and _________ added
A _______ is captured on _______ and _______ is performed
An ________ when a dNTP is added to the DNA, decreasing the ________
__ is detected in each of the wells by _________, allowing the determination of ____________ with no signal when ________
DNA fragments (~200 bp), adapters
Single DNA molecule, a bead, emulsion PCR
H+ ion is released, pH in the well
pH, ION sensitive transistors, whether a dNTP is incorporated, no dNTP is added
Disadvanteges to sanger
requires purified DNA as template
it is difficult to scale up and sequence many templates at the same time for electrophoresis
cost is high
SGS technology disadvantages
The template preparation has a bias in template representation, and procedures can be technically difficult
Shorter reads (signal depends on the average of many molecules, dephasing decreases the read length)
More errors
Use wash and scan method (pausing at each step)
Third generation sequencing technologies
Sequencing by ________________
_________ sequencing by ___________
_______ sequencing (__________)
Sequencing hybridization/ligation(complete genomics)
Single molecule sequencing by synthesis
Nanopore sequencing (nucleotides driven through nanopores, differences in conductance can identify molecules)
Steps in Template Preparation in Complete Genomics
Obtain ____________
Insert ____________
From a __________, make a ____________ containing ________________ using __________ though “_________”; the synthesized molecule is called ________
[Obtain] DNA fragments (400-500 bases)
[Insert] 4 distinct adapter sequences by restriction digest and ligation (circularize the fragments by ligation)
[From a] circular Dna, [make a] long single stranded dna, [containing] many copies of the genomic DNA fragment [using] phi29 DNA polymerase [through] “rolling circle replication” [the synthesized molecule is called] DNA nanoball
Restirction endonucleases are classified based on
subunit composition, co-factor requirements, and DNA-cleavage properties
Type II restriction endonucleases
recognize/cleave DNA at the same site
TYPE IIS restriction endonucleases
Asymmetric recognition w/ cutting at a defined distance
Type III restriction endonucleases
Have Assymetric recognition sites and cut approximately 26 bases away form teh recognition sequence
Steps in modifying and fragment sizing the DNA
Get DNA fragments of 400-500 bases,
- add adaptors 1 and circularize
- perform restriction digest, cut 13 bp to right
- add adapters 2 and & circularize
- restriction digest, cut 13 bp to the left
- add Adaptors 3 and circularize
- perform a restriction digest which cuts 26 bp to both the left and right
- add adaptors 4 & circularize
How to prepare a dna nanoball Start with \_\_\_\_\_\_\_\_ make a \_\_\_\_\_\_\_\_\_\_\_\_ containing \_\_\_\_\_\_\_\_ through \_\_\_\_\_\_\_\_\_ The synthesized molecule is called a
Circular DNA Long single stranded dna many copies of the genomic DNA fragment containing phi29 DNA polymerases "rolling circle replication" DNA nanoball
Principles for sequencing by hybridization/ligation
An __________ binds to ________
Use a ___________ [to] _________ ([each position has] __________
)
a probe that has an _________
- [The hybridized probe is] ______________
Only ______ will stay on template at higher temperature
The label on the probe can tell ______________
This process repeated with ____________
Template DNA has adaptor sequence
- [An] anchor primer oligo [binds to] the adaptor
- [Use a] series of short probes [to] probe the region to be sequenced ([each position has] 4 probes with different fluorescent labels)
- [a probe that has an} exact matching nucleotide hybridizes to template ( and is the only one which does so)
- [The hybridized probe is] ligated to the anchor
- [only] ligated probe [stays on template at higher temperature]
- [The label on the probe can tell] the specific nucleotide at the interrogative position on the template
- [this process repeated with] each cycle interrogating the position on the template
complete genomics advantages
Hight throughput
Low reagent cost
High Accuracy
